Bacteria, fungi and plants recognize and respond to a multitude of environmental, metabolic and cell cycle signals through two-component signal transduction system and phosphorelays. These systems regulate a variety of genes and operons ranging from essential functions and virulence determinants to development and the ripening of fruit. Signal input activates an ATP-dependent autophosphorylation of a histidine residue of a kinase and signal propagation in these systems involves His-Asp phosphotransfer discovered, that of sporulation in bacteria, has been the subject of extensive genetic and biochemical studies. Structural analysis of the components of the pathway have shown how these signaling proteins interact and have given a first view of what residues determine specificity of molecular recognition. The objectives of the present proposal are to elucidate in detail the nature of molecular recognition, molecular specificity, and the mechanism of phosphotransfer in the phosphorelay using crystallographic techniques in conjunction with biochemical and genetic analyses. Two areas of focus will be the structure of the ultimate transcription factor, Spo0A, and its interaction with DNA and the structure of the signal-sensing histidine kinase that regulates phosphoryl input in the phosphorelay. The objective of this latter aim is to understand the mechanism by which signal ligand interaction activates kinase activity. Through these studies the complete mechanism of the pathway of signal transduction from signal ligand binding to gene activation will be understood.